This project includes the development of assays to phenocopy inherited genetic mutations leveraging disease knowledge from basic and clinical research programs with recent advances in molecular biology (e.g., application of TALEN and CRISPR-mediated genome editing). The assay designs are considered in the context of analysis and progression strategies for evaluation of approved drugs and investigational agents using high throughput screening technologies. The lab has a strong emphasis on methods development research to advance assay and screening efficiency in drug discovery and chemical genomics. Peroxisome biogenesis disorders (PBDs): AMP deaminase (AMPD) deficiency: In collaboration with K. Nagaraju (Children's National Medical Center) the ADST laboratory is developing a coincidence reporter assay to aid in the discovery of compounds that increase expression of the enzyme AMP deaminase implicated in the disease idiopathic inflammatory myositis (IIM). IIM is a rare autoimmune progressive disease that afflicts the skeletal muscle of patients. IIM is comprised of errors in both immune regulation and intrinsic muscle metabolism. AMP deaminase (AMPD) catalyzes the deamination of AMP to IMP. In humans AMPD activity is encoded by at least three genes. AMPD isoforms have tissue specific expression patterns in adults and stage specific expression patterns during the muscle development. Drugs which stimulate the expression of AMPD1 are hypothesized to reverse muscle weakness. Diamond Blackfan Anemia (DBA): In collaboration with D. Bodine (NHGRI, NIH) and G. Thomas (U. Cincinnati) the ADST laboratory is developing assay strategies as part of a translational research project for DBA a rare, congenital disease seen in all ethnic groups with a frequency of 7 cases per million live births. The diagnosis is usually made in the 1st yr of life, as a severe anemia that requires transfusion. Unlike other anemias, DBA patients have no reticulocytes in peripheral blood indicating a failure of erythropoiesis whereas production of the white cells and platelets are unaffected. For a small minority of patients, bone marrow transplantation from a healthy sibling is a curative therapy. For patients without a suitable donor a variety of palliative therapies that can prolong life into the third decade. Current treatments include corticosteroid therapy, which results in reduced growth and other complications, or lifelong transfusion and iron chelation. Discovery of Drugs for inherited rare blinding retinal degenerations: Donald J. Zack, M.D., Ph.D., a Johns Hopkins researcher funded by the Foundation Fighting Blindness, is collaborating with Dr. Ingleses ADST laboratory in a public-private partnership aimed at alleviating and curing blinding retinal degenerative diseases. Together they are utilizing ADSTs expertise in assay development, chemical biology and quantitative high-throughput screening (qHTS) to develop assays and chemical library testing paradigms to identify drugs and compounds that have the potential to save and restore vision for people affected by devastating retinal diseases such as retinitis pigmentosa and age-related macular degeneration, the leading cause of blindness in Americans over 50 years of age. The use of primary rodent retinal cells, a limiting cell type typical for typical HTS experiments, benefits from the low-volume assay technology employed in the Inglese laboratory at NCATS. Development of a cell-based high throughput quantitative microscopy-based assay for glucose-regulating multienzyme compartment: In collaboration with S. An (UMBC) the ADST laboratory of NCATS is developing a 1536-well plate format high throughput quantitative microscopy-based assay for discovery of compounds that modulate the formation of a dynamically assembled multienzyme complex regulating glucose-derived carbon flux in cells. Given that glucose metabolism is the central metabolic pathway that balances the cellular needs for both energy and building blocks, the pilot screening of pharmacologically active small molecules for or against the assembly will assist to understand the biological significance of the assembly in the cell. In turn, extensive high throughput screening is anticipated to discover novel pharmacophores, which promote or disrupt the assembly in human disease models, for therapeutic intervention in the treatment of glucose metabolism-associated human diseases. Exploration of novel sources of chemical libraries for antimalarial activity: To probe the biological activity of the stereochemical and structural complexity of novel academic chemical libraries and the chemical diversity of natural products extracts (NPEs) the ADST laboratory is testing the antimalarial potential of these chemical repositories. In a multi-investigator collaboration effort with D. Sherman (U. Michigan), J. Clardy (HMS), J. Porco (BU), S. Schaus (BU), A. Beeler (BU), J.K. Snyder (BU), X.-z., Su (NIAID, NIH), the ADST laboratory has determined EC50s for Plasmodium falciparum viability for each of 2,070 members of the Boston University CMLD compound collection and >16,00 NPEs assembled at the University of Michigan using quantitative high-throughput screening across five parasite lines of distinct geographic origin. From the BU library three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified. From the NPE library a novel recently discovered compound, called Actinoramide A was found to display potent antimalarial activity. The molecular basis for the activity of these compounds is being investigated. Assays for the identification of small molecules up-regulating Scn1a gene expression. Dravet syndrome, a severe epileptic disorder, is caused by nonsense mutations in the SCN1A gene resulting in a non-functional protein. In collaboration with V. Broccoli (Institute of Neuroscience, Milan) we are developing mouse ES cells, capable of differentiation to neurons, containing a coincidence reporter biocircuit edited into the genetic locus encoding the alpha subunit of the neuronal voltage-gated sodium channel. Small molecules that up-regulate the unaffected allele of SCN1A may provide a compensatory mechanism for disease treatment. Controlling cellular senescence with small molecule compounds modulating the 133p53 isoform: In collaboration with C. Harris (NCI) the ADST laboratory of NCATS is developing a 1536-well plate format high throughput laser-cytometry based assay for discovery of compounds that modulate the cellular distribution and half-life of specific p53 isoform in cells. The demonstrated functional and regulatory role served by 133p53 between cellular senescence and autophagy is relevant to both aging and cancer. Compounds effective in modulating cellular phenotypes based on expression of a GFP- 133p53 sensor will be explored for their pharmacological effect in relevant aging and cancer model systems.